Anti-MDM2 single-chain antibody and application thereof

Abstract

The invention discloses an anti-MDM2 single-chain antibody and application thereof, specifically, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises three CDRs, H-CDR1, H-CDR2 and H-CDR3, and the light chain variable region comprises three CDRs, L-CDR1, L-CDR2 and L-CDR3; the amino acid sequences of the H-CDR1, the H-CDR2 and the H-CDR3 are CDR1, CDR2 and CDR3 of a heavy chain variable region as shown in any one of the description; the amino acid sequences of the L-CDR1, the L-CDR2 and the L-CDR3 are CDR1, CDR2 and CDR3 of a light chain variable region as shown in any one of the description.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to an anti-MDM2 single-chain antibody and its applications. Background Technology

[0002] MDM2 (mouse two-microsome homolog 2), a key intracellular E3 ubiquitin ligase, plays a central role in the negative feedback regulation of the p53 signaling pathway. Under DNA damage stress, kinases such as ATM and CHK2 are activated, directly phosphorylating specific sites on MDM2, altering its conformation or protein-protein interaction, weakening the binding of MDM2 to p53, thereby stabilizing p53 and activating its transcriptional function. This regulatory mechanism ensures that cells can rapidly initiate p53-mediated DNA repair or apoptosis programs when facing genotoxic threats. In addition to directly regulating p53, MDM2 can also form homo / heterodimers with MDMX, significantly enhancing the inhibitory effect on p53. MDM2 gene amplification or protein overexpression has become a common mechanism in tumorigenesis. It is estimated that approximately 7-10% of tumors have MDM2 gene amplification, while in tumors such as soft tissue sarcoma, osteosarcoma, and glioblastoma, this proportion can be as high as 20-30%. MDM2 has attracted widespread attention as an important target for cancer therapy. Currently, hundreds of small molecule inhibitors have been evaluated in preclinical studies and many molecules have been tested in clinical trials, but there are currently no FDA-approved MDM2 inhibitors on the market.

[0003] Single-chain variable fragments (ScFvs) represent a significant advancement in the development of genetically engineered antibody technology. Through ingenious molecular design, they utilize a flexible short peptide linker of 15-20 amino acids to connect the antibody's heavy chain variable region (VH) and light chain variable region (VL) end-to-end, thus constructing a complete antigen-binding unit. This design cleverly mimics the antigen-binding pocket formed by the non-covalent interaction of VH and VL in natural antibodies, allowing it to fully retain its specific antigen recognition capability. Due to its small molecular weight, ScFvs exhibit excellent tissue penetration, effectively penetrating structurally abnormal, high-osmolarity blood vessel walls and dense tumor matrix within solid tumors, reaching deep lesion areas where traditional intact antibodies struggle to accumulate effectively. Furthermore, the smaller size of ScFvs results in faster blood clearance and a shorter half-life. Meanwhile, from the perspective of molecular construction, the coding gene of ScFv has a simple structure, is easy to manipulate and modify, and can be fused with various functional effector molecules through mature genetic engineering technology to construct a multifunctional targeted fusion protein. This provides a broad technical platform for the development of a new generation of efficient and precise biological targeted drugs.

[0004] MDM2 functions through a large, flat protein-protein interaction (PPI) interface. Small molecule inhibitors often struggle to completely disrupt or effectively mimic these complex PPIs and are prone to developing resistance due to mutations at their binding sites. Developing novel MDM2 ScFV targeting modules not only holds promise for overcoming the limitations of small molecule ligands but also holds immense potential for applications in MDM2 E3 ligase function, tumor imaging, and protein-targeted drug development. Summary of the Invention

[0005] The purpose of this invention is to provide a single-chain antibody for the MDM2 protein developed using rabbit monoclonal antibody preparation technology and a screening method thereof.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to an MDM2 protein, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three CDRs, H-CDR1, H-CDR2, and H-CDR3, and the light chain variable region comprising three CDRs, L-CDR1, L-CDR2, and L-CDR3; the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are as follows. The CDR1, CDR2, and CDR3 of any of the heavy chain variable regions shown; the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are as follows CDR1, CDR2, and CDR3 of any of the light chain variable regions shown.

[0008] In some implementations, the CDRs of the light chain variable region and the heavy chain variable region are selected from any of the following:

[0009] (a) The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ.ID.NO:7; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ.ID.NO:13;

[0010] (b) The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ.ID.NO:8; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ.ID.NO14;

[0011] (c) The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ.ID.NO:9; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ.ID.NO:15;

[0012] (d) The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ.ID.NO:10; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ.ID.NO:16.

[0013] (e) The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ.ID.NO:11; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ.ID.NO:17.

[0014] (f) The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ.ID.NO:12; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ.ID.NO:18.

[0015] In some implementations, the CDRs of the light chain variable region and the heavy chain variable region are selected from any of the following:

[0016] (A) The amino acid sequence of H-CDR1 is TNTMS (SEQ ID NO:22), the amino acid sequence of H-CDR2 is TINSAGDNAWYASWVNG (SEQ ID NO:23), and the amino acid sequence of H-CDR3 is YAGSSYYFSI (SEQ ID NO:24); the amino acid sequence of L-CDR1 is TLSSAHSTYTID (SEQ ID NO:25), the amino acid sequence of L-CDR2 is LKSDGSYTRGT (SEQ ID NO:26), and the amino acid sequence of L-CDR3 is GADYSGGYV (SEQ ID NO:27);

[0017] (B) The amino acid sequence of H-CDR1 is SYNMA (SEQ ID NO:28), the amino acid sequence of H-CDR2 is WINTGGRAYYASWAKG (SEQ ID NO:29), and the amino acid sequence of H-CDR3 is HSGYTTGFDI (SEQ ID NO:30); the amino acid sequence of L-CDR1 is QASENIYSFLA (SEQ ID NO:31), the amino acid sequence of L-CDR2 is DASTLAS (SEQ ID NO:32), and the amino acid sequence of L-CDR3 is QQGATWSNVDNT (SEQ ID NO:33);

[0018] (C) The amino acid sequence of H-CDR1 is SYGVI (SEQ ID NO:34), the amino acid sequence of H-CDR2 is YINYGGSAYYASWAKS (SEQ ID NO:35), and the amino acid sequence of H-CDR3 is ANGGINRASDI (SEQ ID NO:36); the amino acid sequence of L-CDR1 is QASQSISDYLA (SEQ ID NO:37), the amino acid sequence of L-CDR2 is EASTLTS (SEQ ID NO:38), and the amino acid sequence of L-CDR3 is QSTYYGDNFVPFNA (SEQ ID NO:39).

[0019] (D) The amino acid sequence of H-CDR1 is SYHMI (SEQ ID NO:40), the amino acid sequence of H-CDR2 is AITGSGRTWYASWAKG (SEQ ID NO:41), and the amino acid sequence of H-CDR3 is ETVYSADNI (SEQ ID NO:42); the amino acid sequence of L-CDR1 is TLRTGYSVGEYPLV (SEQ ID NO:43), the amino acid sequence of L-CDR2 is YHTEEFKHQGS (SEQ ID NO:44), and the amino acid sequence of L-CDR3 is AMGSV (SEQ ID NO:45).

[0020] (E) The amino acid sequence of H-CDR1 is VYEVN (SEQ ID NO:46), the amino acid sequence of H-CDR2 is YIYPNGNTYYASWAKG (SEQ ID NO:47), and the amino acid sequence of H-CDR3 is GYDNVNHDKLDI (SEQ ID NO:48); the amino acid sequence of L-CDR1 is TLRTDYSVGEYGLV (SEQ ID NO:49), the amino acid sequence of L-CDR2 is YHTEELKHQGS (SEQ ID NO:50), and the amino acid sequence of L-CDR3 is FTAHATETSLHYV (SEQ ID NO:51);

[0021] (F) The amino acid sequence of H-CDR1 is SYAIS (SEQ ID NO:52), the amino acid sequence of H-CDR2 is IISSGSTYYASWVNG (SEQ ID NO:53), and the amino acid sequence of H-CDR3 is EKDDTTYISGYDPFDP (SEQ ID NO:54); the amino acid sequence of L-CDR1 is TLRTGYSVGEYPLV (SEQ ID NO:55), the amino acid sequence of L-CDR2 is YHTEEFKHQGS (SEQ ID NO:56), and the amino acid sequence of L-CDR3 is VIADATESSLHYV (SEQ ID NO:57).

[0022] In some embodiments, the antibody or its antigen-binding fragment comprises, for example: Any of the amino acid sequences shown.

[0023] In some embodiments, the antibody or its antigen-binding fragment further includes a linker variable region; the heavy chain variable region and the light chain variable region are linked by the linker variable region.

[0024] In some embodiments, the antibody is a single-chain antibody. In some embodiments, the single-chain antibody has a structural composition comprising a VH fragment, a variable linker region, and a VL fragment.

[0025] In some embodiments, the single-chain antibody is prepared via prokaryotic or eukaryotic expression, and the target expression product obtained using this system is a fusion protein; the fusion protein includes the single-chain antibody, or the fusion protein is composed of the single-chain antibody. In some embodiments, the fusion protein expression includes a His tag, meaning the fusion protein can be a fusion protein that fuses an expression tag and the single-chain antibody.

[0026] In some embodiments, the linker variable region is any linker amino acid sequence; as a preferred embodiment, the amino acid sequence of the linker variable region is GGGGSGGGGSGGGGS (SEQ ID NO:58).

[0027] In some implementations, the affinity constant (KD) of the antibody is less than 3 μM.

[0028] In some implementations, the sequence information of the heavy chain variable region and the light chain variable region of the antibody is obtained using rabbit monoclonal antibody preparation technology.

[0029] A second aspect of the present invention provides a nucleic acid molecule or a carrier containing said nucleic acid molecule, said nucleic acid molecule encoding the antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0030] In some embodiments, the vector comprises a recombinant plasmid. In some embodiments, the recombinant plasmid comprises an initial plasmid backbone and a gene linked to the backbone encoding the aforementioned antibody or its antigen-binding fragment.

[0031] In some embodiments, the initial plasmid is a eukaryotic expression plasmid. As a preferred embodiment, the initial plasmid is pCDNA3.4.

[0032] A third aspect of the present invention provides cells containing the nucleic acid molecules or vectors described in the second aspect of the present invention.

[0033] A fourth aspect of the present invention provides a fusion protein containing the antibody or its antigen-binding fragment described in the first aspect of the present invention.

[0034] A fifth aspect of the present invention provides a reagent or kit comprising the antibody or antigen-binding fragment thereof described in the first aspect of the present invention. In some embodiments, the reagent or kit is used to detect MDM2 or MDM2 / P53 protein.

[0035] In this invention, the MDM2 / P53 protein is a complex of MDM2 and P53 proteins.

[0036] In some embodiments, the reagent or kit further comprises a functional unit; in some embodiments, the functional unit includes an MDM2 single-chain antibody heavy chain fragment, an MDM2 single-chain antibody light chain fragment, an MDM2 single-chain antibody mutant, or a tagged MDM2 single-chain antibody, etc.

[0037] In some embodiments, the reagents or kits include, but are not limited to, fluorescence imaging reagents or kits, radiographic diagnostic reagents or kits, nuclear magnetic resonance ELISA reagents or kits, and Western blotting reagents or kits.

[0038] The sixth aspect of the present invention provides a pharmaceutical preparation comprising the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, and a pharmaceutical carrier.

[0039] In this invention, the drug carrier includes, but is not limited to, polymers, liposomes, or nanoparticles.

[0040] The seventh aspect of the present invention provides a method for detecting MDM2 protein for non-diagnostic purposes, which involves co-incubating the antibody or its antigen-binding fragment described in the first aspect of the present invention with the sample to be tested.

[0041] In some embodiments, the method involves utilizing the reactivity of the single-chain antibody with the MDM2 protein and using ELISA or SPR to detect the human MDM2 protein.

[0042] The eighth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention or the pharmaceutical preparation described in the sixth aspect of the present invention in any of the following:

[0043] (1) Use in the preparation of medicaments for the prevention or treatment of diseases associated with abnormal MDM2 protein levels or P53 mutations;

[0044] (2) Prepare products for detecting MDM2 or diagnosing diseases related to abnormal MDM2 protein levels.

[0045] In this invention, the products include, but are not limited to, reagents or kits.

[0046] In this invention, the diseases associated with abnormal MDM2 protein levels include malignant tumors, non-tumor diseases, hereditary diseases, or tissue damage caused by radiotherapy and chemotherapy.

[0047] In this invention, the malignant tumors include, but are not limited to, soft tissue sarcoma, glioblastoma, gastrointestinal stromal tumor, lung cancer, bladder cancer, colorectal cancer, breast cancer, or prostate cancer;

[0048] In this invention, the non-tumor diseases mentioned include, but are not limited to, atherosclerosis, human papillomavirus (HPV) infection, Alzheimer's disease, Parkinson's disease, Huntington's disease, cerebral ischemia, or stroke;

[0049] In this invention, the hereditary diseases include, but are not limited to, Li-Fraumeni syndrome, hereditary breast cancer, hereditary ovarian cancer syndrome, or familial adenomatous polyposis.

[0050] In this invention, the tissue damage caused by radiotherapy and chemotherapy includes, but is not limited to, bone marrow damage or gastrointestinal mucosal damage.

[0051] A ninth aspect of the present invention provides a method for screening antibodies that specifically bind to the MDM2 protein, comprising the following steps:

[0052] i. Antigen design: plasmid design, prokaryotic protein expression and purification, mammalian transient transtransfer protein expression and purification;

[0053] ii. Rabbit immunization: Rabbits were immunized multiple times with antigens. PBMCs and spleen lymphocytes of the immunized rabbits were isolated, RNA was extracted, and cDNA was prepared. The method of constructing single-chain antibody libraries was determined based on the serum titer evaluation results.

[0054] iii. Phage display library construction: Using the immunized cDNA, the scFv gene fragment was amplified, and an M13 single-stranded filamentous phage display antibody immune library was constructed;

[0055] iv. Phage library screening: In vitro targeted screening using MDM2 immunogen;

[0056] v. Sequencing and Phage ELISA assays were used to determine the amino acid sequence of single-chain antibodies with high affinity.

[0057] In some implementations, the phage library construction can be optimized by altering immune cDNA or other methods.

[0058] In some implementations, the specificity and binding affinity of the screening can be improved by adjusting parameters such as the amount of multi-tag protein, the amount of target protein, the incubation time, and the elution conditions.

[0059] A ninth aspect of the present invention provides a method for preparing an antibody as described above, comprising the following steps:

[0060] A recombinant plasmid encoding the aforementioned antibody was prepared at the endotoxin-free transfection level. After transfection with HEK293F, cells were collected. After cell lysis, the secretory supernatant, lysis supernatant, and lysis precipitate were sent for Western blotting. The intracellular supernatant and / or precipitate expressing the target protein were purified by Ni column chromatography, and Western blotting was used to verify whether the target protein was obtained.

[0061] The tenth aspect of the present invention provides a method for treating or preventing diseases associated with abnormal MDM2 protein levels, the method comprising administering an appropriate amount of the aforementioned antibody or pharmaceutical preparation to a subject.

[0062] The beneficial effects of this invention are:

[0063] This invention provides a single-chain antibody against MDM2 protein developed using rabbit immune screening technology. These antibodies possess the ability to specifically recognize MDM2 protein. The developed double-chain antibody can serve as a potential functional unit for biological research and drug development, used to study the key role of MDM2 protein in biological systems and MDM2 / p53 protein-protein interactions, and to deeply analyze the function of the MDM2 E3 ligase. This is of great significance for the prevention and treatment of diseases related to abnormal MDM2 protein expression. Compared with small molecule inhibitors and traditional antibodies, it has the following advantages:

[0064] 1. High specificity: The single-chain antibody provided by this invention binds to the MDM2 protein with high affinity. During the screening process, experimental conditions can be controlled to ensure the obtained single-chain antibody has high specificity, reducing the impact on non-target proteins.

[0065] 2. Good safety profile: Compared with traditional antibodies and small molecule drugs, single-chain antibodies have lower immunogenicity and better biocompatibility, and fewer side effects.

[0066] 3. Wide range of applications: The single-chain antibody provided by this invention can be used to prepare MDM2 and MDM2 / P53 protein detection reagents (kits), study the biological processes of MDM2-related signaling pathways, monitor the dynamic changes of MDM2 protein, identify MDM2 / P53 protein-protein interactions, and diagnose and treat diseases related to abnormal activation of MDM2.

[0067] The single-chain antibodies described in this invention can serve as tools for MDM2 protein biology research, contributing to a deeper understanding of its function and regulatory mechanisms. Single-chain antibodies can also be applied to the treatment of tumors, metabolic disorders, and neurodegenerative diseases. MDM2 is associated with numerous cancers; developing specific single-chain antibodies can disrupt the interaction between MDM2 and p53, thereby regulating signaling pathways and inhibiting tumor growth and spread. Research on MDM2 single-chain antibodies can provide novel drug development directions for these diseases. Attached Figure Description

[0068] Figure 1 Western blot analysis for identifying MDM2 protein expression in mammals;

[0069] Figure 2 Western blot analysis was used to identify and analyze the purified protein.

[0070] Figure 3For the detection of antiserum titer 7 days after 4 immunizations;

[0071] Figure 4 To verify the binding affinity of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 for ELISA;

[0072] Figure 5 To verify the binding affinity of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 for ELISA;

[0073] Figure 6 Verify the binding strength of SEQ ID 1 for SPR;

[0074] Figure 7 Verify the binding strength of SEQ ID 2 for SPR;

[0075] Figure 8 To verify the binding force of SEQ ID 3 for SPR. Detailed Implementation

[0076] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0077] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0078] Unless otherwise stated, the experimental methods, detection methods, and preparation methods not described in detail in this invention all adopt conventional techniques in this technical field.

[0079] Example 1: Prokaryotic expression of MDM2 protein

[0080] According to databases such as NCBI, PDB, and UniProt, the theoretical molecular weight of the MDM2 protein is 55.23 kDa (excluding the tag). The amino acid sequence translation is as follows:

[0081] MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVS ENRCHLEGGSDQKDLVQELQEEKPSSSHLVSRPSTSSRRRAISETEENSDELSGERQRKRHKSDSISLSFDESLALCVIREICCERSSSSESTGTPSNPDLDAGVSEHSGDWLDQDSVSDQFSV EFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAGESDTDSFEEDPEISLADYWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEKAKLENSTQAEEGFDVPDCKKTIVNDSR ESCVEENDDKITQASQSQESEDYSQPSTSSSIIYSSQEDVKEFEREETQDKEESVESSLPLNAIEEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKKRNKPCPVCRQPIQMIVLTYFP (SEQ ID NO:19)

[0082] Build Option 1:

[0083] The protein was constructed into the pCzn1 vector and expressed via N-His tag fusion. The protein molecular weight was 56.66 kd (including the tag).

[0084] MNHKVHHHHHHMCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQES SDSGTSVSENRCHLEGGSDQKDLVQELQEEKPSSSHLVSRPSTSSRRRAISETEENSDELSGERQRKRHKSDSISLSFDESLALCVIREICCERSSSSESTGTPSNPDLDAGVSEHSGDWLDQDSVS DQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAGESDTDSFEEDPEISLADYWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEKAKLENSTQAEEGFDVPDCKKTIVN DSRESCVEENDDKITQASQSQESEDYSQPSTSSSIIYSSQEDVKEFEREETQDKEESVESSLPLNAIEEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKKRNKPCPVCRQPIQMIVLTYFP (SEQ ID NO:20)

[0085] Construction Method Two:

[0086] The protein was constructed into the pSumo-mut vector and seamlessly cloned after the glycine residue following the Sumo tag. The molecular weight of the protein was 69.4 kDa (including the tag).

[0087] (SEQ ID NO:21)

[0088] pSumo-mut-MDM2 and pCZN1-MDM2 vectors were transformed into E. coli Arctic-Express and BL21(DE3):

[0089] (1) Add 1 μL of plasmid to 100 μL of competent bacteria and place on ice for 20 min.

[0090] (2) Heat shock for 90 seconds, then immediately place on ice for 5 minutes, and add 600 μL of LB culture medium.

[0091] (3) Shake at 220 r / min for 1 h, centrifuge, and then spread the entire mixture onto LB agar plates containing 50 μg / mL Amp / Kan. Invert and incubate overnight.

[0092] IPTG induces the expression of the pSumo-mut-MDM2 and pCZN1-MDM2 vector fusion proteins:

[0093] (1) Pick a single clone from the transformation plate and inoculate it into a test tube containing 3 mL LB medium containing 50 μg / mL Amp / Kan. Shake overnight at 220 r / min.

[0094] (2) The next day, the inoculum was diluted 1:100 in 30 mL of LB medium containing 50 μg / mL Amp / Kan. Shake at 220 r / min until the cells are formed It is 0.6-0.8.

[0095] (3) Take out 1 mL of culture, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet with 100 μL 1× loading buffer.

[0096] (4) Add IPTG to the remaining culture until the final concentration is 0.2 mM. Shake at 220 r / min overnight to induce fusion protein expression.

[0097] (5) Take 1 mL of culture, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet in 100 μL of 1× loading buffer. Centrifuge the remaining culture at 4000 r / min for 10 min, discard the supernatant, and resuspend the bacterial pellet in PBS; after sonicating the resuspended solution, take the supernatant and the pellet solution respectively and add them to the loading buffer for resuspending.

[0098] Ni-column affinity purification of fusion proteins:

[0099] (1) Using a low-pressure chromatography system, the supernatant was loaded onto a Ni-IDA-Sepharose Cl-6B affinity chromatography column pre-equilibrated with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min.

[0100] (2) Rinse with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min until the outflow is complete. The value reaches the baseline.

[0101] (3) Rinse with Ni-IDA Washing-Buffer (20 mM Tris-HCl, 30 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 mL / min until the outflow is complete. The value reaches the baseline.

[0102] (4) Elute the target protein with Ni-IDA Elution-Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 mL / min and collect the eluent.

[0103] (5) The protein solution collected above was added to a dialysis bag and dialyzed overnight with PBS.

[0104] (6) Perform 12% SDS-PAGE analysis.

[0105] Example 2 MDM2 protein expression in mammals

[0106] Plasmid extraction:

[0107] (1) Take 400 mL of overnight cultured bacterial solution, centrifuge at 9000 r / min for 10 min, drain the supernatant as much as possible, and collect the bacterial cells.

[0108] (2) Resuspend the bacterial precipitate in 25 mL of solution P1 and vortex until completely suspended.

[0109] (3) Add 25 mL of solution P2, gently invert the container 4-7 times to fully lyse the cells, and let it stand at room temperature for 4 min.

[0110] (4) Add 25 mL of solution P3, and immediately gently invert the tube 4-7 times to mix thoroughly. A white flocculent precipitate will appear. Let it stand on ice for 3-5 min, centrifuge at 13000 r / min for 10 min, and carefully transfer the supernatant to a 50 mL centrifuge tube. Pre-cool the tube on ice.

[0111] (5) Add 0.1 times the volume of ice-cooled endotoxin scavenger to the supernatant obtained in the previous step, invert and rotate to mix 7-10 times, and ice bath for 10 minutes, inverting and mixing occasionally.

[0112] (6) Water bath, inverted and mixed thoroughly Incubate for 20-30 minutes.

[0113] (7) Centrifuge at 12000 r / min for 10 min at room temperature to separate phases.

[0114] (8) The solution separates into two phases. The upper aqueous phase contains DNA, and the lower oily phase contains endotoxin. Transfer the DNA-containing upper aqueous phase to a new tube and discard the oily layer.

[0115] (9) Add 0.5 times the volume of binding liquid PB to the upper aqueous phase obtained in the previous step, mix thoroughly, transfer to the adsorption column (place the adsorption column in the collection tube), centrifuge at 13000 r / min for 30-60 s, and discard the waste liquid in the collection tube.

[0116] (10) Add rinsing solution, centrifuge at 13000 r / min for 30-60 s, and discard the waste liquid.

[0117] (11) Place the adsorption column back into the empty collection tube, centrifuge at 13000 r / min for 2 min, and remove the washing solution.

[0118] (12) Remove the adsorption column and place it in a centrifuge tube. Add 500 μL of elution buffer to the middle of the adsorption membrane.

[0119] Let stand at room temperature for 2 min, then centrifuge at 13000 r / min for 1 min.

[0120] Transfection:

[0121] (1) Culture HEK293 cells to ensure they are in the logarithmic growth phase with a viability greater than 95%. cell / mL.

[0122] (2) PEI needs to be prepared in advance Preheating: Preheat DNA to room temperature for 10 minutes beforehand. Transfection buffer. Preheat.

[0123] (3) Transfection system

[0124]

[0125] (4) Add the required plasmid and PEI to the transfection buffer, mix them together thoroughly, and incubate at 37°C for 15 min.

[0126] (5) Add the incubated plasmid to the cells. Suspension culture.

[0127] (6) Supplement N source and growth factor 24 h after transfection.

[0128] (7) Count the cells on day 6, observe the cell status and mortality rate, and harvest the cells.

[0129] (8) 5000 r / min, 15 min, collect cell supernatant and precipitate for downstream identification and purification.

[0130] Cell disruption:

[0131] (1) Add an appropriate volume of lysis buffer (50 mM Tris, 300 mM NaCl, 20 mM Midazole, pH 8.0) to the collected cells, and then sonicate to completely resuspend the cells.

[0132] (2) Cell ultrasound parameters: power: 250 W; time: 6 s for over 6 seconds and 3 s for 3 minutes.

[0133] (3) Centrifuge at 12000 r / min for 15 min and collect the precipitate.

[0134] (4) Add an appropriate volume of dissolution buffer (50 mM Tris, 300 mM NaCl, 8 M MUrea, 20 mM Imidazole, pH 8.0) to the collected precipitate, and then sonicate to completely resuspend the cells.

[0135] (5) Cell ultrasound parameters: power: 250 W; time: 6 s for 3 s for 3 min.

[0136] (6) Centrifuge at 12000 r / min for 15 min and collect the supernatant.

[0137] Protein purification by precipitation and fragmentation:

[0138] (1) Using a low-pressure chromatography system, the mixture of secretion supernatant and Ni column incubation was slowly added to the purification empty column.

[0139] (2) Equilibrate the Ni column with Balance Buffer (50 mM Tris, 300 mM NaCl, 8 M Urea, 20 mM Imidazole, pH 8.0) at a flow rate of 0.5 mL / min until the eluent is obtained. The value reaches the baseline.

[0140] (3) The target protein was eluted with Elution Buffer (50 mM Tris, 300 mM NaCl, 8 M Urea, 500 mM Midazole, pH 8.0) at a flow rate of 1 mL / min, and the effluent was collected for electrophoresis detection.

[0141] Western Blot analysis:

[0142] (1) Load 10 μL of sample.

[0143] (2) After the sample loading is completed, run the polyacrylamide gel at 100 V to complete the stacking gel, and then increase the voltage to 120 V until the electrophoresis is completed.

[0144] (3) After electrophoresis, remove the gel and transfer the membrane at a constant voltage of 100 V for about 1.5 h.

[0145] (4) After electroporation, remove the membrane and wash it four times with PBS for 5 minutes each time. Then place it in 5% PBS.

[0146] Sealing in milk powder sealing solution 1 h.

[0147] (5) Dilute the primary antibody with blocking buffer, and place the membrane in the primary antibody dilution buffer. The reaction lasted for 1 hour.

[0148] (6) Wash the membrane 4 times, 5 min each time; dilute the secondary antibody with blocking buffer containing 5% milk. The membrane in the secondary antibody... The reaction lasted for 1 hour.

[0149] (7) Washing and developing ECL.

[0150] Western blotting results are as follows: Figure 1 As shown in the figure. The results indicate that the target protein is expressed in the intracellular precipitate. The intracellular precipitate was further selected as the sample for Ni column purification, and the purification results are shown in the figure. Figure 2 As shown, the target protein was obtained after purification.

[0151] Example 3 Identification of MDM2 protein

[0152] Proteolytic digestion:

[0153] (1) Gel cutting: Cut the protein strips and place them into an EP tube;

[0154] (2) Washing: Add MilliQ water and wash for 1 min, centrifuge to remove supernatant, and wash twice with water;

[0155] (3) Decolorization: Add 50% MeOH / 50mM Decolorize in a 37℃ constant temperature oven for 30 minutes, then centrifuge to remove the supernatant;

[0156] (4) Dehydration: Add 100% ACN, shake for 30 seconds until the colloidal particles turn white, then remove the liquid;

[0157] (5) Alkylation: Add 25 mM DTT / 50 mM DTT to the dried granules. The reaction was carried out at 56 degrees Celsius for 30 minutes. DTT was then removed, and 55 mM IAA / 50 mM IAA was added. React at room temperature in the dark for 30 minutes;

[0158] (6) Washing: Extract the IAA and wash 3 times with MilliQ water;

[0159] (7) Dehydration: Dehydrate 100% ACN until the particles turn white;

[0160] (8) Enzyme digestion: Dilute trypsin to 20 ng / µl with 25 mM NH4HCO3; add an appropriate amount of trypsin to each tube and incubate on ice for 30 min; then add an appropriate amount of 25 mM NH4HCO3. Cover the gel particles and incubate overnight at 37 degrees Celsius for enzyme digestion.

[0161] (9) Peptide extraction: Place the entire EP tube box into an ultrasonic instrument and sonicate for 15-20 minutes to extract the peptides into a new EP tube.

[0162] Peptide purification ZipTip

[0163] (1) Wetting the column: Absorb 10µl of 100% ACN, discard, and repeat twice;

[0164] (2) Acidification of the column: Immerse 10 µl of 0.1% TFA, discard, and repeat twice;

[0165] (3) Adsorption of samples: blow and aspirate the sample 15 times;

[0166] (4) Remove impurities: Aspirate 10 µl of 0.1% TFA, discard, and repeat twice;

[0167] (5) Elution of sample: Aspirate 10 µl of 0.1% TFA-, 700% ACN and transfer this eluent into a new EP tube;

[0168] (6) Vacuum drying.

[0169] Mass spectrometry detection:

[0170] (1) The peptide fragments were dissolved in the sample dissolving solution (0.1% formic acid, 2% acetonitrile) at 13200 rpm. Centrifuge for 20 min, collect the supernatant, and perform mass spectrometry identification;

[0171] (2) Liquid phase parameters

[0172] (a) Column information:

[0173] 300 um idx 5mm, Acclaim PepMap RSLC C18, 5 um, (Thermo, 160454)

[0174] Acclaim PepMap 75um X 150mm, C18, 3um, 100A (Thermo, 160321)

[0175] (b) Mobile phase information

[0176] Mobile phase A: 0.1% formic acid

[0177] Mobile phase B: 0.1% formic acid, 80% ACN

[0178] Flow rate: 300 nL / min

[0179] (c) Analysis time: 65 min

[0180] Effective gradient: Phase B increases from 5% to 90%.

[0181] As shown in Table 1.

[0182] Table 1

[0183]

[0184] (3) Mass spectrometry parameters

[0185] The separated peptides were directly analyzed online using a Thermo Scientific Q Exactive mass spectrometer, with the following parameters:

[0186] (a) Primary mass spectrometry parameters

[0187] Resolution: 70,000

[0188] AGC target: 3e6

[0189] Maximum IT: 40 ms

[0190] Scan range: 350 to 1800 m / z

[0191] (b) Secondary mass spectrometry parameters

[0192] Resolution: 17,500

[0193] AGC target: 1e5

[0194] Maximum IT: 60 ms

[0195] TopN: 20

[0196] NCE / stepped NCE: 27.

[0197] Example 4: Preparation of Rabbit Monoclonal Antibody

[0198] Animal immunization: Two New Zealand rabbits were immunized with the protein antigen MDM2 obtained in Example 1, 150 μg / rabbit, once every 2 weeks, for a total of 5 immunizations.

[0199] Antiserum titer test:

[0200] The antiserum titer was detected by indirect ELISA. The specific steps of indirect ELISA are as follows:

[0201] (1) Dilute the protein antigen MDM2 to the required concentration with PBS coating buffer, mix well, and add 100 μL to each well of the strip. Refrigerate overnight. Coating antigen: protein antigen MDM2; coating concentration: 5 μg / mL, 100 μL / well; coating buffer: phosphate buffer (PBS, pH 7.4).

[0202] (2) After coating, discard the coating solution, wash the plate 3 times, and add 200 μL of blocking solution to each well. Incubate for 1 hour. Remove the microplate, discard the internal solution, and wash the plate once.

[0203] (3) Antiserum was diluted 3 times at a ratio of 1:500, with 100 μL per well. Incubator for 1 hour.

[0204] (4) Remove the microplate, discard the internal liquid, wash the plate 3 times, and add 100 μL of diluted enzyme-labeled secondary antibody to each well. Enzyme-labeled secondary antibody: goat anti-rabbit, 1:50,000. Incubator for 1 hour.

[0205] (5) Remove the microplate, discard the internal solution, wash the plate 4 times, and add 100 μL of TMB chromogenic solution to each well. 15 min.

[0206] (6) Add 100 μL of 1M HCl solution to each well to stop the reaction. Immediately take a reading at 450 nm on the microplate reader. The dilution corresponding to the well with an OD value that is 2.1 times greater than the set negative control OD value is defined as the potency of the sample.

[0207] After four immunizations, the antiserum titer of peripheral blood from New Zealand rabbits was measured 7 days later. Figure 3 As shown, both rabbits produced a good immune response after immunization. (Number) and Rabbit antiserum titers against MDM2 protein antigen were all greater than 1093.5K times dilution in ELISA.

[0208] Example 5: Construction of Antibody Development Library

[0209] The library was constructed using the M13 phage display system, which consists of the pCantab5E phage vector, E. coli TG1, and M13KO7 helper phages.

[0210] Preparation of ScFV library fragments:

[0211] Using cDNA as a template, the ScFV antibody fragment was amplified using primers. An appropriate amount of PCR product was subjected to 1% agarose gel electrophoresis. The PCR product was loaded onto an agarose gel for electrophoretic separation, and the target fragment was purified and recovered using a gel extraction kit. The recovered gel product was used as the template for the first round of VH / VL PCR, and amplification was performed using the first round template to obtain VH / VL with added linker. The recovered gel product was used as the second round linker-VH and VL-linker; the second round VH / VL was recombined into ScFV, and the recovered product became the third round PCR product; the third round was used as the template to amplify ScFV with added restriction enzyme sites. The DNA fragment of the library obtained by PCR amplification was recovered and purified, then digested with NotI-HF / Sfi1, purified, recovered, quantified, aliquoted, and stored for subsequent experiments.

[0212] Electroconversion and library construction:

[0213] The digested vector was ligated with the ScFV fragment. The ligation product was purified and electroporated into E. coli TG1, yielding 15 mL of transformation product. 10 μL (i.e., [the product]) was then taken. Perform a series of 10-fold serial dilutions and take... Three gradients of ampicillin-resistant plate counting were performed to assess library volume, calculated as: clone number × dilution factor × total volume of transformation products (ml). The remaining transformation products were plated onto ampicillin-resistant plates and incubated overnight. The following day, the plate was scraped off, mixed thoroughly, and then 20% glycerol was added to the final concentration. The plates were then aliquoted and stored at -70°C.

[0214] The digested vector was ligated with the ScFV fragment. The ligation product was purified and electroporated into E. coli TG1 to obtain 10 mL of transformation product. 10 μL (i.e., [the product]) was then taken. Perform a series of 10-fold serial dilutions (ml), and take... Three gradients of ampicillin-resistant plate counting were performed to assess library capacity, calculated as: clone number × dilution factor × total volume of transformation products (ml). Remaining transformation products were plated onto ampicillin-resistant plates and incubated overnight. The following day, the plate was scraped off, mixed thoroughly, and then 20% glycerol was added to the final concentration. The plates were aliquoted and stored at -80°C. Thirty clones were randomly selected for colony PCR analysis.

[0215] Immunotherapy library quality analysis:

[0216] Thirty single clones were randomly selected from the serially diluted plates of the library and sequenced using the forward sequencing primer pcantab5E. A total of 28 valid sequencing clones were obtained (clones 14 and 15 were terminated early). The amino acid sequence of the ScFV fragment was compared and analyzed. 28 of the 30 clones contained antibody fragments with the correct reading frame.

[0217] The overall accuracy of the library is approximately 28 ÷ 30 × 100% = 93.3%. A Neighbor-Joining Tree for the ScFV fragments was constructed using MEGA software, and the results show that the ScFV fragments exhibit good diversity. In conclusion, the quality of the ScFV library meets the expected requirements.

[0218] Example 6: Screening of Phage Antibody Library

[0219] Screening: A rabbit monoclonal antibody library displaying MDM2 was used for in vitro targeted screening against the MDM2 immunogen. Results are shown in Table 2.

[0220] Table 2 (1)

[0221]

[0222] Table 2 (2)

[0223]

[0224] Preparation of phage supernatant:

[0225] (1) Pick 500 single colonies from the plates from the first and second rounds of delivery and put them into 1 mL of 2× YT-AI-hp medium. Shake overnight at 37°C and 200 rpm.

[0226] (2) Centrifuge at 5000 rpm for 10 min. Carefully aspirate 600 μL of the supernatant into a 1.5 mL centrifuge tube, which is the sample to be tested for Phage ELISA.

[0227] Example 7: ELISA screening results

[0228] Phage ELISA detection:

[0229] Coating: MDM2, 0.1 μg / mL, coated, overnight at 4℃.

[0230] Sealing: 2% skim milk powder, 200 μL / well, 37℃, 1h.

[0231] Primary antibody: Sample, 100 μL / well, 37℃, 1h.

[0232] Secondary antibody: anti-M13 antibody (HRP), 1:25000, 100 μL per well. 37℃, 1h.

[0233] Color development: 100 μL of colorimetric reagent per well. 37℃, 15 min.

[0234] Termination: Termination solution, 100 μL / well.

[0235] Reading board: Antibody titer.

[0236] Soluble ELISA test:

[0237] I. Preparation of soluble proteins in the periplasmic cavity:

[0238] Five unique clones (in E. coli TG1) selected by phage display were induced to express by IPTG at 37°C. After centrifugation, the bacterial cells were collected and soluble proteins were extracted from the periplasmic cavity.

[0239] (1) Take 10 μL from each tube of monoclonal bacterial culture and add it to 1 mL of 2×YT-AG (ampicillin 100 µg / ml). -1 (with 2% glucose), incubated overnight at 37°C and 220 rpm.

[0240] (2) Take 0.4 mL of overnight cultured monoclonal bacterial culture into 20 mL of 2×YT-AG (0.1% glucose), incubate at 37℃ and 220 rpm for about 2.5 h until the logarithmic growth phase.

[0241] (3) Centrifuge at 3000 rpm for 15 min and discard the supernatant. Add 20 mL of 2×YT-AI (IPTG final concentration of 1 mM), incubate overnight at 30°C and 200 rpm.

[0242] (4) Centrifuge at 3000 rpm for 15 min, discard the supernatant, resuspend the precipitate with 0.5 mL of pre-cooled 1×TES, place on ice for 15 min, then add 0.75 mL of pre-cooled 1 / 5 × TES and mix thoroughly. Place on ice for 30 min.

[0243] (5) Centrifuge at 13,000 rpm, 4 °C, for 10 min, and carefully transfer the supernatant to a new centrifuge tube, which is the peritoneal supernatant.

[0244] II. Soluble ELISA assay (coated with MDM2)

[0245] Starting with the original solution, the supernatant was diluted 5 times, resulting in 5 gradients. 100 μL of each supernatant was used to detect ELISA wells coated with MDM2 antigen (0.1 μg / mL / well). 1×PBS was used as a negative control.

[0246] (1) Coating: MDM2 antigen, 0.1 μg / mL, 100 μL / well, overnight.

[0247] (2) Sealing: 2% skim milk powder, 200 μL / well, 37℃, 1h.

[0248] (3) Primary antibody: Soluble sample, starting with stock solution, 5-fold dilution, 100 μL / well, 37℃, 1h. PBS as control.

[0249] (4) Secondary antibody: E-tag Antibody [HRP], pAb, Rabbit, 1:2000, 100 μL / well. 37℃, 1h.

[0250] (5) Color development: 100 μl of color development solution per well, 37℃, 15 min.

[0251] (6) Termination: 100 μl of termination solution per well

[0252] After routine screening, Phage ELISA yielded 33 positive clones, but all reacted with the multi-tag protein. Therefore, further optimization of conditions was performed, including increasing the number of routine screening rounds, expanding the screening sample size, increasing the coating concentration, and increasing the pre-adsorption time. As shown in Table 3, after four screenings, five weakly positive clones meeting the criteria were obtained. Further increasing the coating concentration and pre-adsorption time, a fifth screening was conducted, yielding two more positive clones that met the requirements and showed weak positive results by Soluble ELISA (as shown in Table 4).

[0253] Table 3. Results of Soluble ELISA assay after the fourth round of selection

[0254]

[0255] Table 4. Results of Soluble ELISA assay after the fifth round of selection

[0256]

[0257] Further sequencing was performed, and the results are as follows.

[0258] The amino acid sequences of the variable regions of the heavy chain are shown in SEQ. IN. NO. 7-12:

[0259] SEQ. IN. NO. 7:

[0260] WPSVEESGGRLVTPGTPLTLTCTVSGIALSTNTMSWVRQAPGEGLEWIGTINSAGDNAWYASWVNGRFTISKTSTTVDLKMTSLTIEDTATYFCARYAGSSYYFSIWGPGTLVTVSS

[0261] SEQ. IN. NO. 8:

[0262] TVSGFTISSYNMAWVRQAPGEGLECTGWINTGGRAYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARHSGYTTGFDIWGPGTLVTISS

[0263] SEQ. IN.NO.9:

[0264] PVVGGVRGGLFKPTDTLTLTCTVSGFSLSSYGVIWVRQAPGKELEWIGYINYGGSAYYASWAKSRSTITRNTNENTVTLKMTSLTGADTATYFCARANGGINRASDIWGPGTLVTVSS

[0265] SEQ. IN.NO.10:

[0266] PVGGGVQGRLVTPGTPLTLTCTVSGFSLSSYHMIWVRQAPGKGLEWIGAITGSGRTWYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARETVYSADNIWGPGTLVTISS

[0267] SEQ. IN.NO.11:

[0268] QSLEESGGRLVTPGTPLTLTCTVSGFSLSVYEVNWVRQAPGKGLEWIGYIYPNGNTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCSRGYDNVNHDKLDIWGQGTLVTISS

[0269] SEQ. IN.NO.12:

[0270] SRWRRSGGRLVTPGTPLTLTCTVSGFSLSSYAISWVRQAPGKGLEWIGIISSSGSTYYASWVNGRFTISKTSTTVDLKITSPTTEDTATYFCAREKDDTTYISGYDPFDPWGPGTLVTISS

[0271] The amino acid sequences of the light chain variable regions are shown in SEQ. IN.NO.13 - 18. <00!​​​ELVLTQSPSVSAALGASAKLTCTLSSAHSTYTIDWYQQKPGTAPRYLVQLKSDGSYTRGTGVPDRSSGSSSGADRYLIISSVQADDEADYYCGADYSGGYVFGGGTQLTVT

[0274] SEQ. IN.NO.14:

[0275] LDLTQTPSPVSAAVGDTVTINCQASENIYSFLAWYQQKPGHPPKLLIYDASTLASGVPSRFKGSGSGTQFTLTISGVECADAATYYCQQGATWSNVDNTFGGGTELEIL

[0276] SEQ. IN.NO.15:

[0277] ELVLTQTPASVSEPVGGTVTIKCQASQSISDYLAWYQQKPGQPPKLLIYEASTLTSGVPSRFSGGGSGTEYTLTISDLECADAATYYCQSTYYGDNFVPFNAFGGGTELEIL

[0278] SEQ. IN.NO.16:

[0279] QPVLTQSLSLSASLGTTARLTCTLRTGYSVGEYPLVWLQQVPGRPPMYLLTYHTEEFKHQGSGVHSRFSGSKDTSENAGVLSISGLQPEDEANYYCAMGSVFGGGTQLTVT

[0280] SEQ. IN.NO.17:

[0281] ELVLTQSPSLSASLGTTARLTCTLRTDYSVGEYGLVWLQQVPGRPPRYLLTYHTEELKHQGSGVHSRFSGSKDTSENAGVLSISGLQPEDEANYYCFTAHATETSLHYVFGGGTQLTVT

[0282] SEQ. IN.NO.18:

[0283] ELVLTQSPSLSASLGTTARLTCTLRTGYSVGEYPLVWLQQVPGRPPMYLLTYHTEEFKHQGSGVRSRFSGSKDTSENAGVLSISGLQPEDEANYYCVIADATESSLHYVFGGGTQLTVT

[0284] The amino acid sequences of the single-chain antibodies are shown in SEQ.ID.NO.1-6:

[0285] SEQ. IN.NO.1:

[0286] WPSVEESGGRLVTPGTPLTLTCTVSGIALSTNTMSWVRQAPGEGLEWIGTINSAGDNAWYASWVNGRFTISKTSTTVDLKMTSLTIEDTATYFCARYAGSSYYFSIWGPGTLVTVSSGGGGSGGGGSGGGGSELVLTQSPSVSAALGASAKLTCTLSSAHSTYTIDWYQQKPGTAPRYLVQLKSDGSYTRGTGVPDRSSGSSSGADRYLIISSVQADDEADYYCGADYSGGYVFGGGTQLTVT

[0287] SEQ. IN.NO.2:

[0288] TVSGFTISSYNMAWVRQAPGEGLECTGWINTGGRAYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARHSGYTTGFDIWGPGTLVTISSGGGGSGGGGSGGGGSLDLTQTPSPVSAAVGDTVTINCQASENIYSFLAWYQQKPGHPPKLLIYDASTLASGVPSRFKGSGSGTQFTLTISGVECADAATYYCQQGATWSNVDNTFGGGTELEIL

[0289] SEQ. IN.NO.3:

[0290] PVVGGVRGGLFKPTDTLTLTCTVSGFSLSSYGVIWVRQAPGKELEWIGYINYGGSAYYASWAKSRSTITRNTNENTVTLKMTSLTGADTATYFCARANGGINRASDIWGPGTLVTVSSGGGGSGGGGSGGGGSELVLTQTPASVSEPVGGTVTIKCQASQSISDYLAWYQQKPGQPPKLLIYEASTLTSGVPSRFSGGGSGTEYTLTISDLECADAATYYCQSTYYGDNFVPFNAFGGGTELEIL

[0291] SEQ. IN.NO.4:

[0292] PVGGGVQGRLVTPGTPLTLTCTVSGFSLSSYHMIWVRQAPGKGLEWIGAITGSGRTWYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARETVYSADNIWGPGTLVTISSGGGGSGGGGSGGGGSQPVLTQSLSLSASLGTTARLTCTLRTGYSVGEYPLVWLQQVPGRPPMYLLTYHTEEFKHQGSGVHSRFSGSKDTSENAGVLSISGLQPEDEANYYCAMGSVFGGGTQLTVT

[0293] SEQ. IN.NO.5:

[0294] QSLEESGGRLVTPGTPLTLTCTVSGFSLSVYEVNWVRQAPGKGLEWIGYIYPNGNTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCSRGYDNVNHDKLDIWGQGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSLSASLGTTARLTCTLRTDYSVGEYGLVWLQQVPGRPPRYLLTYHTEELKHQGSGVHSRFSGSKDTSENAGVLSISGLQPEDEANYYCFTAHATETSLHYVFGGGTQLTVT

[0295] SEQ. IN.NO.6:

[0296] SRWRRSGGRLVTPGTPLTLTCTVSGFSLSSYAISWVRQAPGKGLEWIGIISSSGSTYYASWVNGRFTISKTSTTVDLKITSPTTEDTATYFCAREKDDTTYISGYDPFDPWGPGTLVTISSGGGGSG GGGSGGGGSELVLTQSPSLASLGTTARLTCTLRTGYSVGEYPLVWLQQVPGRPPMYLLTYHTEEFKHQGSGVRSRFSGSKDTSENAGVLSISGLQPEDEANYYCVIADATESSLHYVFGGGTQLTVT

[0297] Example 8 Single-chain antibody expression

[0298] Plasmid extraction:

[0299] (1) Take 400 mL of overnight cultured bacterial solution, centrifuge at 9000 r / min for 10 min, drain the supernatant as much as possible, and collect the bacterial cells.

[0300] (2) Resuspend the bacterial precipitate in 25 mL of solution P1 and vortex until completely suspended.

[0301] (3) Add 25 mL of solution P2, gently invert the container 4-7 times to fully lyse the cells, and let it stand at room temperature for 4 min.

[0302] (4) Add 25 mL of solution P3, and immediately gently invert the tube 4-7 times to mix thoroughly. A white flocculent precipitate will appear. Let it stand on ice for 3-5 min, centrifuge at 13000 r / min for 10 min, and carefully transfer the supernatant to a 50 mL centrifuge tube. Pre-cool the tube on ice.

[0303] (5) Add 0.1 times the volume of ice-cooled endotoxin scavenger to the supernatant obtained in the previous step, invert and rotate to mix 7-10 times, and ice bath for 10 minutes, inverting and mixing occasionally.

[0304] (6) Incubate in a 42℃ water bath, invert and mix well, then incubate at 42℃ for 20-30 min.

[0305] (7) Centrifuge at 12000 r / min for 10 min at room temperature to separate phases.

[0306] (8) The solution separates into two phases. The upper aqueous phase contains DNA, and the lower oily phase contains endotoxin. Transfer the DNA-containing upper aqueous phase to a new tube and discard the oily layer.

[0307] (9) Add 0.5 times the volume of binding liquid PB to the upper aqueous phase obtained in the previous step, mix thoroughly, transfer to the adsorption column (place the adsorption column in the collection tube), centrifuge at 13000 r / min for 30-60 s, and discard the waste liquid in the collection tube.

[0308] (10) Add rinsing solution, centrifuge at 13000 r / min for 30-60 s, and discard the waste liquid.

[0309] (11) Place the adsorption column back into the empty collection tube, centrifuge at 13000 r / min for 2 min, and remove the washing solution.

[0310] (12) Take out the adsorption column, put it into a centrifuge tube, add 500 μL of elution buffer to the middle part of the adsorption membrane, place it at room temperature for 2 min, and centrifuge at 13000 r / min for 1 min.

[0311] Transfection:

[0312] (1) Culture HEK293F cells into the logarithmic growth phase, with a viability greater than 95%, and the cells are at a growth rate of 1.8-2.2 × 10⁻⁶. 6 cell / mL.

[0313] (2) PEI needs to be preheated to 37°C in advance, DNA needs to be preheated to room temperature for 10 minutes in advance, and transfection buffer needs to be preheated to 37°C.

[0314] (3) Transfection system

[0315] The transfection system is shown in Table 5.

[0316] Table 5

[0317]

[0318] (4) Add the required plasmid and PEI to the transfection buffer, mix them together thoroughly, and incubate at 37°C for 15 min.

[0319] (5) Add the incubated plasmid to the cells and culture in suspension at 37°C.

[0320] (6) Supplement N source and growth factor 24 hours after transfection.

[0321] (7) Count the cells on day 6, observe the cell status and mortality rate, and harvest the cells.

[0322] (8) 5000 r / min, 15 min, collect cell supernatant and precipitate for downstream identification and purification.

[0323] Cell disruption:

[0324] (1) Add an appropriate volume of lysis buffer (50 mM Tris, 300 mM NaCl, 20 mM Midazole, pH 8.0) to the collected cells, completely resuspend the cells, and then sonicate them.

[0325] (2) Cell ultrasound parameters:

[0326] Power: 250 W

[0327] Time: 3 seconds pause after 6 seconds, total 3 minutes.

[0328] (3) Centrifuge at 12000 r / min for 15 min and collect the supernatant and precipitate.

[0329] (4) Add an appropriate volume of dissolution buffer (50mM Tris, 300mM NaCl, 8M Urea, 20mM Imidazole, pH 8.0) to the collected precipitate, and then sonicate to completely resuspend the cells.

[0330] (5) Cell ultrasound parameters:

[0331] Power: 250 W

[0332] Time: 3 seconds pause after 6 seconds, total 3 minutes.

[0333] (6) Centrifuge at 12000 r / min for 15 min and collect the supernatant.

[0334] Cell lysis supernatant protein purification

[0335] (1) Using a low-pressure chromatography system, the mixture of sample and Ni column after incubation is slowly added to the purification empty column.

[0336] (2) Equilibrate the Ni column with Balance Buffer (50mM Tris, 300mM NaCl, 20mM Imidazole, pH8.0) at a flow rate of 0.5 mL / min until the OD280 value of the effluent reaches the baseline.

[0337] (3) Elute the target protein with Washing Buffer (50 mM imidazole, 50 mM Tris, 300 mM NaCl, pH 8.0) at a flow rate of 1 mL / min and collect the eluent.

[0338] (4) Elute the target protein with Elution Buffer (500mM imidazole, 50mM Tris, 300mM NaCl, pH 8.0) at a flow rate of 1 mL / min and collect the eluent.

[0339] Cell lysis, precipitation, and purification

[0340] (1) Using a low-pressure chromatography system, the mixture of sample and Ni column after incubation is slowly added to the purification empty column.

[0341] (2) Equilibrate the Ni column with Balance Buffer (50mM Tris, 300mM NaCl, 20mM Imidazole, 8M Urea, pH8.0) at a flow rate of 0.5 mL / min until the OD280 value of the effluent reaches the baseline.

[0342] (3) Elute the target protein with Washing Buffer (50 mM imidazole, 50 mM Tris, 300 mM NaCl, 8 M Urea, pH 8.0) at a flow rate of 1 mL / min and collect the eluent.

[0343] (4) Elute the target protein with Elution Buffer (500mM imidazole, 50mM Tris, 300mM NaCl, 8M Urea, pH8.0) at a flow rate of 1 mL / min and collect the eluent.

[0344] Western blot analysis

[0345] (1) Load 10 μL of sample.

[0346] (2) After the sample loading is completed, the polyacrylamide gel is run at 100 V to complete the stacking gel, and then the voltage is increased to 120 V.

[0347] Until the electrophoresis is finished.

[0348] (3) After electrophoresis, remove the gel and transfer the membrane at a constant voltage of 100 V for about 1.5 h.

[0349] (4) After electroporation, remove the membrane and wash it four times with PBS for 5 minutes each time. Then place it in 5% PBS.

[0350] The milk powder was sealed in a sealing solution at 37°C for 1 hour.

[0351] (5) Dilute the primary antibody with blocking solution, and react the membrane in the primary antibody dilution solution at 37°C for 1 h.

[0352] (6) Wash the membrane 4 times, 5 min each time; dilute the secondary antibody with blocking buffer containing 5% milk. Incubate the membrane with the secondary antibody at 37℃.

[0353] The reaction lasted for 1 hour.

[0354] (7) Washing and developing ECL.

[0355] Some single-chain antibodies were obtained through eukaryotic expression.

[0356] Example 9: ELISA test for binding strength

[0357] The purified protein was validated using ELISA to test the binding ability of the single-chain antibody to MDM2. The specific procedure is as follows:

[0358] 1. Coating: 6 SCFV antibodies, 5 μg / mL, coated overnight at 4℃.

[0359] 2. Sealing: 2% skim milk powder, 200 μL / well, 37℃, 1h.

[0360] 3. Antigen: MDM2-Biotin, starting at 5 μg / mL, 2-fold dilution, 100 μl / well, react at 37℃ for 1 h.

[0361] 4. Secondary antibody: SA-HRP, 1:10,000, 100 μL / well. React at 37℃ for 1 h.

[0362] 5. Color development: 100 μL of colorimetric solution per well. Incubate at 37°C for 15 min.

[0363] 6. Termination: Termination solution, 100 μL / well.

[0364] 7. Read the board: Antibody titer.

[0365] like Figure 4 , Figure 5 As shown, the single-chain antibodies SEQ.ID.NO: 1-6 obtained through screening can all bind to MDM2.

[0366] Example 10 SPR test bonding strength

[0367] SPR assays were performed using a Biacore 8K instrument. The target protein (MDM2, 20 μg / ml, pH 5.5) was immobilized on a CM5 chip, and the run buffer was HBS-EP (0.01M HEPES, 0.15M NaCl, 3 mM EDTA, 0.5% P2O, pH 7.4). The fusion protein was diluted to different gradients using HBS-EP. Flow rate: 30 μL / min, binding time: 120 s, dissociation time: 60 s. Analysis was performed using Biacore Insight Evaluation software.

[0368] like Figure 6 , Figure 7 , Figure 8 The single-chain antibody described in the embodiments has a K-type effect on MDM2. D All were below 3 μM.

[0369] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment specifically binding to an MDM2 protein, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising three CDRs, H-CDR1, H-CDR2 and H-CDR3, and said light chain variable region comprising three CDRs, L-CDR1, L-CDR2 and L-CDR3; characterized in that, The amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are as follows: The CDR1, CDR2, and CDR3 of any of the heavy chain variable regions shown; the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are as follows CDR1, CDR2, and CDR3 of any of the light chain variable regions shown.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The CDRs of the light chain variable region and the heavy chain variable region are selected from any of the following: (a) The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ.ID.NO:7; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ.ID.NO:13; (b) The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ.ID.NO:8; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ.ID.NO14; (c) The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ.ID.NO:9; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ.ID.NO:15; (d) The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ.ID.NO:10; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ.ID.NO:

16. (e) The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ.ID.NO:11; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ.ID.NO:

17. (f) The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ.ID.NO:12; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ.ID.NO:

18.

3. A nucleic acid molecule or a carrier containing said nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or its antigen-binding fragment as described in claim 1 or 2.

4. A cell containing the nucleic acid molecule or vector as described in claim 3.

5. A fusion protein, characterized in that, The fusion protein contains the antibody or its antigen-binding fragment as described in claim 1 or 2.

6. A reagent or kit, characterized in that, The reagent or kit described herein contains the antibody or antigen-binding fragment thereof as described in claim 1 or 2.

7. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the antibody or its antigen-binding fragment as described in claim 1 or 2, and a pharmaceutical carrier.

8. A method for detecting MDM2 protein for non-diagnostic purposes, characterized in that, The antibody or its antigen-binding fragment as described in claim 1 or 2 is used to co-incubate with the sample to be tested.

9. The use of the antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, or the pharmaceutical preparation as claimed in claim 7, in any of the following: (1) Use in the preparation of medicaments for the prevention or treatment of diseases associated with abnormal MDM2 protein levels; (2) Prepare products for detecting MDM2 protein or diagnosing diseases related to abnormal MDM2 protein levels.

10. The application according to claim 9, characterized in that, The diseases associated with abnormal MDM2 protein levels include malignant tumors, non-tumor diseases, genetic diseases, or tissue damage caused by radiotherapy and chemotherapy; The malignant tumors mentioned include soft tissue sarcoma, glioblastoma, gastrointestinal stromal tumor, lung cancer, bladder cancer, colorectal cancer, breast cancer, or prostate cancer; The non-tumor diseases mentioned include atherosclerosis, human papillomavirus infection, Alzheimer's disease, Parkinson's disease, Huntington's disease, cerebral ischemia, or stroke; The hereditary diseases mentioned include Li-Fraumeni syndrome, hereditary breast cancer, hereditary ovarian cancer syndrome, or familial adenomatous polyposis. The tissue damage caused by radiotherapy and chemotherapy includes bone marrow damage or gastrointestinal mucosal damage.

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